The present invention relates to a novel antiviral agent from the plant Pongamia pinnata and a process for the preparation of the said agent. The antiviral agent is active against White spot syndrome virus (WSSV). Therefore, it is useful for the prophylaxis and for treatment of White spot syndrome virus (WSSV) infected Penaeus monodon (Shrimp). Currently there is no specific drugs or scientifically established treatment methodology available for WSSV infection in shrimp.
The invention also relates to an improved process for the preparation of Bis (2-methyl heptyl) phthalate of the formula (1)

Pongamia pinnata (Linn) Pierre Leguminosae, Papillionaceae, Synonym, Pongamia glabra vent is an indigenous medicinal plant. Pongamia pinnata is commonly called Pongam, Karanj or Karanga in Hindi, is a medium sized glabrous, fast growing and adaptable tree found through out India and distributed from tropical Asia to northern Australia. Pongamia pinnata has soft, shiny, burgundy new leaves, which glow in light. These will age to bright lime green and then to dark green. It is used extensively throughout for its traditional medicine.
Activities of the plant
The aqueous and alcohol extract of the leaves and seed of Pongamia pinnata were shown to exhibit antifilarial activity. Ethanol extract (70%) of Pongamia pinnata leaves shows antinociceptive and antipyretic activity and also anti-inflammatory activity. The seed oil of Pongamia pinnata shows antibacterial activity. The seed extract of Pongamia pinnata shows antiviral activity against Herpes simplex virus.

Antifilarial activity, Antinociceptive, antipyretic and anti-inflammatory activity from the ethanol extract of the leaves of the plant is known. Moreover the leaves of the said plant are known to be hot, digestive, laxative, anthelmintic and cure piles, wounds and other inflammations (Kirtikar and Basu, 1933). A hot infusion of leaves is used as a medicated bath for relieving rheumatic pains and for cleaning ulcers in gonorrhea and scroflilous enlargement (Chopra et al., 1933; Satyavati et al., 1987).
But the antiviral activity of the extract of the leaves of the plant p ongmia pinnata is not hitherto known.
Seeds
The seed and seed oil of this plant have been used for treating various inflammatory and infectious diseases such as leucoderma, leprosy, lumbago, muscular and articular rheumatism (Nadkarni,1954).
In sanskritic India, seeds were used for skin ailments. Powdered seeds are valued as a febrifuge, tonic and in bronchitis and whooping cough. The seeds contain pongam oil, a bitter, red brown, thick, non-drying, nonedible oil, 27-36% by weight, which is used for tanning leather, soap, as a liniment to treat scabies, herpes, and rheumatism and as an illuminating oil (Burkill, 1966). Also used for lubrication and indigenous medicine.
The antiviral activity of the seeds of the plant is also known against Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2).
A crude aqueous seed extract of P. pinnata was reported to completely inhibit the growth of another kind of virus called herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) at concentrations of 1 and 20 mg/ml (w/v), respectively, as shown by complete absence of cytopathic effect (Elanchezhiyan et al 1993).
The antiviral activity of the seeds of the plant is known and it is unknown whether such seed extract would exhibit similar activity against WSSV and needs testing in the WSSV. As it has been known the antiviral activity we have not tested in WSSV.

Wood
The wood is yellowish white, coarse, hard, and beautifully grained, but is not durable. Use of the wood is limited to cabinetmaking, cart wheels, posts, and fuel (Allen and Allen, 1981). The ash of the wood is used in dyeing. The antiviral activity of the wood of the said plant is not known
Ayurvedic medicine described the root and bark as alexipharmic, anthelmintic, and useful in abdominal enlargement, ascites, biliousness, diseases of the eye, skin, and vagina, itch, piles, splenomegaly, tumors, ulcers, and wounds; the sprouts, considered alexeteric, anthelmintic, apertif, and stomachic, for inflammation, piles and skin diseases; the leaves, anthelmintic, digestive, and laxative, for inflammations, piles and wounds; the flowers for biliousness and diabetes; the fruit and seed for keratitis, piles, urinary discharges, and diseases of the brain, eye, head, and skin, the oil for biliousness, eye ailments, itch, leucoderma, rheumatism, skin diseases, worms, and wounds.
A crude aqueous seed extract of the plant P. pinnata was reported to completely inhibit the growth of another kind of virus called Herpes simplex virus type-1 (HSV-1) and tvpe-2 (HSV-2) at concentrations of 1 and 20 mg/ml (w/v), respectively, as shown by complete absence of cytopathic effect (Elanchezhiyan et al 1993).
Yunani use the ash to strengthen the teeth, the seed, carminative and depurative, for chest complaints, chronic fevers, earache, hydrocele, and lumbago; the oil, styptic and vermifuge, for fever, hepatalgia, leprosy, lumbago, piles, scabies, and ulcers.
Chemistry of the Plant
Phytochemical examination shows that Pongamia pinnata (-)Isoglabrachromene, a new natural chromenoflavanone, and hexacosanyl caffeate and triacontanyl caffeate, two new esters, have been isolated together with 11 known flavonoids and betulinic acid from the stem bark of Pongamia glabra. Murari et al 2001

The stem-bark of Pongamia glabra, has given besides the known flavones kanugin and desmethoxy kanugin, a new chromenoflavone pongachromene Mukerjee etal 2001
Isopongaglabol and 6-methoxyisopongaglabol, two new
hydroxyfuranoflavones, together with two furanoflavones 5-methoxyfurano(8,7-4",5")flavone and 5-methoxy-3,'4'-methylenedioxyfurano(8,7-4'',5")flavone, two simple flavones, desmethoxykanugin and fisetin tetramethyl ether, a chromenoflavanone, ovalichromene B, two triterpenes, cycloart-23-ene-3,25-diol and friedelin, and -sitosterol—glucoside were isolated from the petrol and CHCI3 extracts of the flowers of Pongamia glabra. Sunil et al 2001
Pongaglabol, a new hydroxyfuranoflavone, and aurantiamide acetate, a rarely occurring modified phenylalanine dipeptide, have been isolated together with 4 furanoflavones, karanjin, lancheolatin B, kanjone and pinnatin, a simple flavone, kanugin, a chromenoflavanone (-)-isolonchocarpin, two furanodiketones pongamol and ovalitenone, and -sitosterol from the petrol and chloroform extracts of the flowers of Pongamia glabra. Sunil et al 2001
New furanoflavone, glabone, has been isolated and identified as 4'-methoxy-6,7-furanoflavone from the flowers of Pongamia glabra. Das Kanungo 2001.
Isopongachromene, a new chromenoflavone together with karanjin, pongamol,
pongapin, kanjone and pongaglabrone were isolated from the ethanolic extract of the
seed oil of Pongamia glabra. The structure of isopongachromene has been
established as 6-methoxy-6",6"-dimethyl-3',4'-methylenedioxychromeno
(7,8,2",3")flavone on the basis of spectral evidence and confirmed by synthesis. Ved et al 2001
Glabrachalcone, a new chromenochalcone has been isolated along with a known chromenochalcone from an ethanolic extract of the seed oil of Pongamia glabra. The structure of glabrachalcone has been established as 2'-hydroxy-2,4,5-trimethoxy-6",6"-dimethylchromeno(4',3':2",3")chalcone on the basis of spectral evidence and was confirmed by synthesis. Pathak et al 2001

From the seeds of Pongamia glabra, pongaglabrone, a new furano-flavone has been isolated and its structure established as 3\4'-methylenedioxy-furano-(2":3''-7:8)-flavone. R. N. Khanna and T. R. Seshadri 2001
The chemical examination of the galls present on the infected leaves of the plant Pongamia glabra has yielded, in addition to a number of known compounds, two new prenylated -diketones, pongagallone-A and pongagallone-B.
Furanoflavones, furanoflavonols, chromenoflavones, flavones and
furanodiketones are flavonoids from fruits of P.pinnata. The oil has a high content of triglycerides, and its disagreeable taste and odor are due to bitter flavonoid constituents, pongamiin and karanjin.
Flavonoids with an anellated furan ring have diverse biological activities including antibacterial, antifungal, anti tubercular, anti inflammatory, quinone reductase and cytotoxicity. Dihydro furano compounds, furano rotenoids and bis furano flavonoids have anti cancer agent.
Previous studies have reported the presence of following compounds from Pongamia pinnata. They are Lanceolatin, Kanjone, Prongaglabrone, Pongaglabol, Pongaglabol methyl ether, Pongol mrthyl ether, Milleltocalyxin C, Ovalifolin, Pongamoside A, Pongamoside B, Pongamoside C, Pinnatin, Gamatin, Karanjin, Pongapin, Pongamol, Ovalitenone, Ponganone, and Pongrotenone.
Activities of the compounds isolated from various parts of Pongamia pinnata
The compounds that showed anti-herpes simplex virus HSV-1 and HSV-2 are Ovalifolin and Pongol methyl ether . Karanjin is a nitrification inhibitor (Majumdar, 2002), juvenomimetic (Mathur et al.,1990) and synergist to insecticide (Sighamony et al., 1983). Pongamol (Sighamony et al., 1983) and Pongapin (Attri et al., 1973) was also found to be a synergist to insecticide. Karanjin has showed anti-tubercular (suppressing growth of Mycobacterium tuberculosis) (Ramaswamy and Sirsi 1960), anti fungal (Pan et al., 1985) and antibacterial activity. Lanceolatin and Pongaglabol

methyl ether and Pinnatin were reported to be the most active antifungal agent of fliranoflavanoids (Pan et al., 1985). Pongamol has been explored extensively to have sedative and depressant (Mahli et al., 1989). Pongarotene showed antifungal, antibacterial and phytotoxic activities.
White spot disease outbreaks have caused mass mortalities and severe damage to the shrimp culture industry among cultured penaeid shrimp worldwide. To date, about 21 shrimp viruses have been reported. Amongst them WSSV is a major pathogen with high infectivity, high mortality and a broad host range infecting several crustacean species including shrimp, crab and crayfish. Upon infection, WSSV infected cells are observed first in the stomach, gill and cuticular epidermis of the shrimp. It subsequently spreads systematically to other tissues of mesodermal and ectodermal origin. In this context, control of disease is very important to insure the long-term survival of shrimp aquaculture.
At present there is no remedy or treatment for WSSV infection in the culture systems of Penaeid shrimp though Protective effects of various immunostimulants have been reported against WSSV infection, for example, oral administration of peptidoglycans or lipopolysaccharides in P.japonicus (Itami et al., 1998; Takahashi et al., 2000), and glucan in Penaeus monodon (Song et al., 1997; Chang et al., 1999). Oral administration of crude fucoidan (CF) extracted from Sargassum polycystum can reduce the impact of White Spot Syndrome virus (WSSV) infection in Penaeus monodon.
There is no scientifically proven suitable drugs for use in shrimp aquaculture industry is available as on date and therefore a solution particularly a herbal drug of the kind shown in the present investigation to solve the problem of WSSV is considered to be a boon and could be welcome by everyone in the industry.
The main objective of the present invention is to provide a novel antiviral agent from the leaves of the plant Pongamia pinnata, containing Bis (2-methyl heptyl) phthalate of the formula (1)


Another objective of the present invention is to provide a process for the preparation of a novel antiviral agent from the leaves of the plant Pongamia pinnata which is active against White spot syndrome virus (WSSV).
Accordingly the present invention provides a novel extract from the leaves of the plant Pongamia pinata containing as an active ingredient Bis (2-methyl heptyl) phthalate of the formula (1)

According to another embodiment of the present invention there is provided a process for the preparation of a novel antiviral agent which is active against White spot syndrome virus (WSSV) which comprises
(i) shade drying the fresh leaves of Pongamia pinnata and coarsely powdering the
dried leaves,
(ii) Extracting the dry powder with absolute ethanol for a period in the range of 3-5
hours, preferably at 5 hrs at a temperature in the range of 40-50 ° C, preferably at 50
Deg C and
(iii) Concentrating the resulting extract and
(iv) subjecting the extract to PTLC over silica gel G ( Acmes) using ethanol
According to another embodiment of the invention there is provided an improved process for the preparation of bis (2-methyl heptyl) phthalate, which comprises

(i) shade drying the fresh leaves of Pongamia pinnata and coarsely powdering the dried leaves,
(ii) Extracting the dry powder with absolute ethanol for a period in the range of 3-5 hours, preferably for 5 hrs at a temperature in the range of 40-50° C, preferably at 50 Deg C and
(iii) Concentrating the resulting extract (iv) subjecting the extract to PTLC over silica gel G ( Acmes) using ethanol and
(v) extracting the three major bands obtained in step (iv) with boiling chloroform to get bis (2-methyl heptyl) phthalate,
The UV spectrum of the second band from the above chloroform extract showed
peaks at 264nm, which indicated an aromatic ring. The IR spectrum showed no
hydroxyl group (absence of peak around
3400 (m-1)). It showed the presence of ester moiety (1728 and 1273 cm"1) and aromatic system (1631,1460, 745 and 700 cm-1
The mass spectrum (EIMS) gave the molecular ion at m/z 390 corresponding to the structure 1 for the compound. The presence of phthalate was shown by the peak at m/z 166 (100%) [Phthalic acid].
The !HNMR of the compound of the second band from the above chloroform extract) clearly showed the symmetry of the molecule. The two terminal methyls and the two secondary methyls at C-21 appeared at 8 0.82 integrating for 12 protons. The aromatic protons H-3 and H-4 appeared at 8 7.65 and 7.45 as AABB1 system. The methylene protons at H-l1 attached to the ester appeared at 8 4.15 as multiplet. The other methylene protons appeared at 8 1.25. The 13C NMR spectrum also confirmed the structure. The ester carbonyl appeared at 8 167.8 while the methylene attached to ester oxygen appeared 8 68.1. The terminal methyl appeared at 8 14.1 while the methyl

attached to C-2 appeared at 810.9. The three aromatic carbons C-2, C-3 and C-4 appeared at 5 130.9, 128.8 and 132.4 respectively.
The compound of the formula 1 was shown to be bis (2-methyl heptyl) phthalate, which has antiviral properties against WSSV virus that is responsible for devastation of the shrimp industry in the world. Moreover this is the first time that the compound has been isolated and identified from the extract of the leaves of the plant Pongamia pinnata.
The leaves of the plant Pongamia pinnata were collected and a voucher specimen has been deposited in the herbarium.
Antibacterial assay of crude extract of P.pinnata
Vibrio sp., Pseudomonas sp., and Streptococcus sp. were used as test organisms. The antibacterial activity was performed by an agar plate diffusion assay. Thio Sulphate bile salt sucrose agar plates (TCBS) were used for Vibrio sp., Nutrient agar pates (NA) was used for Pseudomonas sp and Tryptic soy agar plates (TSA) was used for Streptococcus sp. The paper discs containing various concentrations of the crude extracts were then applied on the plates. The inoculated plates were incubated at 30°C for 24-48 hrs. The inhibition zones around the discs were measured in mm diameter. The solvent ethanol was used as control.
Antibacterial activity of crude of P.pinnata against Vibrio sp., Pseudomonas sp., and Streptococcus sp. with varying zones of inhibition were noticed. At a concentration of 14mg/ml, the zone of inhibition was 14mm, 21mm and 16mm in diameter for Vibrio sp., Pseudomonas sp., and Streptococcus sp respectively
Minimal inhibitory concentration (MIC) of crude extract of P.pinnata against Vibrio sp., Pseudomonas sp., and Streptococcus sp. The crude extract was diluted in Nutrient broth with different concentration. These were then inoculated with lOO μl of the standard test organism containing 106 CFU/ml with a turbidity of

Mc.Farland No 0.5. tubes were incubated at 30°C for 24-48 hrs. The result was read by visual observation for turbidity in the tubes for growth. MIC was determined as the least concentration of extract inhibiting the growth of the test organism.
The minimum inhibitory concentration (MIC) of crude extract of P.pinnata required to inhibit the growth of Vibrio sp. (lmg/ml), Pseudomonas sp. .(2mg/ml), and Streptococcus sp.(lmg/ml)/. The MIC values revealed that the crude extract was effective against Vibrio sp. Pseudomonas sp, and Streptococcus sp.
Anti-WSSV assay of crude extract of P.pinnata
Healthy shrimp P. monodon weighing 10-12 g were collected from the backwaters of Muttukadu, Chennai. They were maintained in biofiltered laboratory tanks containing aerated seawater at 30°C. The animals were fed with autoclaved commercial pellatized shrimp feed. The shrimp were divided into 3 groups of 15 shrimp each. Crude extract of P.pinnata was fed to the shrimp by mixing it with commercial pellatized shrimp feed. Two different concentrations crude extract of P.pinnata viz. 200 (μg/g Body weight/Day and 300 (μ g/g Body weight/Day of shrimp were used. The crude extract was fed for 4 days before and for 15 days after challenging the shrimp with WSSV by orally feeding them with WSSV infected muscle tissue of P. monodon.. The pelletized feed without crude extract of P.pinnata was given to the control group, which were also challenged with WSSV. The shrimp were monitored for infection and mortality for about 15 days.
Shrimp (15 No.) of 10-12g fed with the pellatized feed containing crude extract of P.pinnata 200 and 300 |μg/g of body weight/day before and then after being experimentally challenged with WSSV, the percentage of survival of P. monodon was observed.
It was found that the group of P monodon experimentally challenged with WSSV which were fed with extract of P.pinnata 200 and 300 μg/g of body weight/day showed 40% and 80% of survival even after 15 days of post infection,

whereas the control shrimp fed without the extract of P.pinnata showed 0% of survival even after 6 days of post infection.
Anti-WSSV assay of TLC fractions of P. pinnata
The shrimp were divided into 4 groups of 10 shrimp each. The compound separated from the crude extract of P.pinnata using TLC was fed to the shrimp by mixing it with commercial pellatized shrimp feed. Three different concentrations of the compound from P.pinnata viz. 50mg/kg Body weight/Day, lOOmg/kg Body weight/Day and 150 mg/kg Body weight/Day were used. The shrimp were monitored for infection and mortality for about 15 days.
The groups of P. monodon experimentally challenged with WSSV were fed with the three different fractions at three different concentration of P.pinnata 50, 100 and 150 mg/Kg of body weight/day. The Ilnd Band showed 60% and 50% of survival even after 15 days of post infection at 100 and 150 mg/Kg of body weight/day. The I and III Band showed very less survival of 30% and 20% after 7 days of post infection at 150 and 100 mg/Kg of body weight/day. Whereas the control shrimp fed without extract of P.pinnata showed 40% of survival 3-5 days after post infection and none of the WSSV infected control shrimp survived after 6 days of infection.
The results of our studies show that the extract contains an antiviral agent which is effective against WSSV virus and further we have identified the active antiviral compound responsible for antiviral activity from the extract of the leaves of
the plant Pongamia pinnata as Bis (2-methyl heptyl) phthalate .
Spectroscopic studies Analytical Material And Methods
The IR spectrum was taken in KBr disc on a Shimadzu FT-IR instrument. The UV-Visible spectrum was taken in ethanol on a Shimadzu UV-1601 instrument. The lH NMR spectrum was recorded in CDCb on a Jeol instrument at 400 MHz while 13C

2. A process for the preparation of a novel extract of the plant Pongamia pinata which
is active against White spot syndrome virus (WSSV) which comprises
(I) shade drying the fresh leaves of Pongamia pinnata and coarsely powdering the
dried leaves,
(ii) Extracting the dry powder with absolute ethanol for a period in the range of 3-5
hours, preferably for 5 hrs at a temperature in the range of 40-50 ° C, preferably at 50
Deg C and
(iii) Concentrating the resulting extract and
(iv) subjecting the extract to PTLC over silica gel G ( Acmes) using ethanol
3. An improved process for the preparation of bis (2-methyl heptyl) phthalate,
which comprises
() shade drying the fresh leaves of Pongamia pinnata and coarsely powdering the dried leaves,
(ii) Extracting the dry powder with absolute ethanol for a period in the range of 3-5 hours, preferably for 5 hrs at a temperature in the range of 40-50 ° C, preferably at 50 DegC (iii) Concentrating the resulting extract

(iv) subjecting the extract to PTLC over silica gel G ( Acmes) using ethanol and
(v) extracting the three major bands obtained with boiling chloroform to obtain bi
(2-methyl heptyl) phthalate,
4. A novel extract from the leaves of the plant Pongamia pinata containing as an active ingredient Bis (2-methyl heptyl) phthalate of the formula (1) bis (2-methyl heptyl) phthalate, substantially as herein described
5. A process for the preparation of a novel extract from the leaves of the plant Pongamia pinata which is active against White spot syndrome virus (WSSV) substantially as herein described
6. An improved process for the preparation of Bis (2-methyl heptyl) phthalate of the formula (1) substantially as herein described